Dr. Guillemin and coworkers at the Salk Institute have recently (Science, 218, 585-587 (Nov. 5, 1982), see also New York Times, Oct. 29, 1982 at page 1, column 2) reported the isolation, synthesis, and biological activity of a group of related substances they have called growth hormone releasing factor (GRF). This factor has been sought after for decades by scientists but such search has been, until now, unrewarding due to the minute quantities in which such substance occurs naturally.
The successful isolation of GRF has been due in part to the discovery of the ectopic production of GRF in large amounts by pancreatic tumors associated with acromegaly. Three forms of GRF derived from the pancreatic tumor have been observed. These forms, consisting of three homologous peptides of 44, 40 and 37 amino acids in length, are identical at the amino terminal and differ in the termination point of the carboxyl terminal. The 44-amino acid GRF is further distinguished in having an amide group at the carboxy terminus whereas the other two forms have a free carboxy group at that terminus. It has been found that removal of the amide group to produce the free acid form of the 44 amino acid GRF results in a significant loss of biological activity.
The amidated form of GRF(1-44) is apparently the parent molecule and has been indicated to possess the highest biological activity in vitro. However, all three peptides have been found to be virtually equally potent in vivo. It has further been shown that the removal of the amino terminal tyrosine from GRF results in complete loss of bioactivity indicating that the active core of the molecule starts with the first amino terminal amino acid.
Rivier and coworkers have recently reported (Nature, 300, 276-278, Nov. 18, 1982) that synthetically produced GRF(1-29)-NH.sub.2, GRF(1-32)-NH.sub.2, GRF(1-39)-NH.sub.2 and GRF(1-40)-NH.sub.2 displayed in vitro biological activity at similar potencies (with a factor of 2) to GRF(1-40)-OH.
Growth in animals is believed to be regulated by a cascade of bio-regulatory molecules. Thus, the hypothalmus produces GRF which in turn acts upon the pituitary to cause release of growth hormone. The pituitary is maintained under negative feedback control by somatostatin and insulin growth factor (IGF). GRF has been found to be enormously active, exhibiting an ED.sub.50 of approximately 50 fmole/ml or 75 pg/ml and has been found to release micrograms/ml levels of growth hormone in the blood. Thus, GRF can be utilized therapeutically in most of the areas now considered candidates for treatment by growth hormone. Examples of such therapeutic uses include the treatment of pituitary dwarfism, diabetes resulting from abnormalities in growth hormone production, enhancement of wound healing, treatment of burns and retardation of the aging process. Due to its favorable potency compared to growth hormone itself, GRF will have major advantages in the agricultural field as well. Agricultural uses would include, for example, stimulating development of fowl or animals raised for meat so as to allow either marketing at an earlier time or else allow the farmer to produce a larger animal per equal time on feed to present methodology. In addition, GRF would be useful in stimulation of milk production in dairy cows and increasing egg production in chickens.
While GRF in its various forms is of a molecular size which would allow for synthesis by either solid phase or solution phase peptide synthetic methods, it is believed that for economic, large scale production of these therapeutically valuable substances the use of recombinant DNA technology is preferred. Using known techniques of DNA recombination, a DNA sequence containing the structural code for GRF could be inserted into a replicable expression vehicle under the control of appropriate control elements including a promoter-operator sequence and a sequence coding for a ribosome binding site. The expression vehicle would then be used to transform a host microorganism, such as a bacterium, which would be grown up and subjected to conditions under which it would express GRF.
Unfortunately, several potential problems exist which hinder the production of GRF by recombinant DNA technology. It has been observed that polypeptides having the molecular size of GRF tend to be more subject to degradation by the proteases which are present in bacteria such as E. coli than are larger protein molecules. Moreover, for reasons which are not fully understood, the cellular machinery which regulates transcription and translation apparently operates more efficiently with longer chain DNA, thus making it difficult to achieve acceptable levels of expression of smaller polypeptides such as GRF. Yet another problem which must be overcome if GRF is to be produced by recombinant DNA technology is that of finding a suitable means of purifying and isolating the GRF from the other bacterial proteins and endotoxins produced by bacterial hosts such as E. coli.
It has been suggested to ligate a DNA sequence encoding the amino acid sequence of GRF with a DNA sequence encoding the amino acid sequence of a larger protein whose amino acid sequence is known and inserting the ligated DNA sequence into an expression vector for the purpose of expressing a fused protein. The fused protein would be much less susceptible to degradation by bacterial proteases than would GRF alone. One could select a fusion protein which is known to be capable of expression at high levels in known expression vectors, for example an interferon protein, and thereby obtain high levels of expression of the fused GRF. Moreover, since there are available monoclonal antibodies which selectively recognize and bind antigenic sites on the interferon protein, one could conveniently purify the fused GRF-interferon protein by passing a crude bacterial extract containing the fusion protein through a column in which the monoclonal antibody is bound to a solid support and then eluting the bound fusion protein from the column using an appropriate elution solvent.
Once the fusion protein was purified, the GRF polypeptide would have to be cleaved from the fused interferon (or other fused protein) and isolated. Cyanogen bromide cleavage is the simplest method by which such cleavage could be effected. Since cyanogen bromide selectively cleaves at the carboxy side of a methionine residue, it would be necessary to construct the ligated DNA sequence by inserting a nucleotide codon for methionine immediately after the codon for the carboxy terminal amino acid residue of the interferon and before the codon for the first amino acid residue of GRF.
The aforementioned strategy for producing GRF by recombinant DNA technology suffers one flaw; to wit, the GRF polypeptide sequence identified by Dr. Guillemin contains a single methionine residue at position 27. Thus, cyanogen bromide cleavage to liberate the GRF from the expressed fusion protein could not be effected without simultaneously cleaving the GRF polypeptide itself. Another, albeit less serious, drawback is that the carboxy-terminus of the GRF produced by recombinant DNA technology would be in the form of an acid, rather than an amide. In the case of the full sequence polypeptide, GRF(1-44), this would mean that an additional and costly amidation step would be necessary to obtain the polypeptide in its fully active form.